Impact of glucose metabolism on PD-L1 expression in sorafenib-resistant hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related mortality worldwide. Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that binds to programmed cell death-1 (PD-1), which is expressed in activated T cells and other immune cells and has been employed in cancer therapy, including HCC. Recently, PD-L1 overexpression has been documented in treatment-resistant cancer cells. Sorafenib is a multikinase inhibitor and the only FDA-approved treatment for advanced HCC. However, several patients exhibit resistance to sorafenib during treatment. This study aimed to assess the effect of glucose deprivation on PD-L1 expression in HCC cells. We used PD-L1-overexpressing HepG2 cells and IFN-γ-treated SK-Hep1 cells to explore the impact of glycolysis on PD-L1 expression. To validate the correlation between PD-L1 expression and glycolysis, we analyzed data from The Cancer Genome Atlas (TCGA) and used immunostaining for HCC tissue analysis. Furthermore, to modulate PD-L1 expression, we treated HepG2, SK-Hep1, and sorafenib-resistant SK-Hep1R cells with rapamycin. Here, we found that glucose deprivation reduced PD-L1 expression in HCC cells. Additionally, TCGA data and immunostaining analyses confirmed a positive correlation between the expression of hexokinase II (HK2), which plays a key role in glucose metabolism, and PD-L1. Notably, rapamycin treatment decreased the expression of PD-L1 and HK2 in both high PD-L1-expressing HCC cells and sorafenib-resistant cells. Our results suggest that the modulation of PD-L1 expression by glucose deprivation may represent a strategy to overcome PD-L1 upregulation in patients with sorafenib-resistant HCC.


Correlation between PD-L1 expression and glycolysis in patients with HCC
The Cancer Genome Atlas (TCGA), using the OncoLnc TCGA data portal (www.oncol nc.org), and immunostaining assays on HCC tissues were used to establish a correlation between PD-L1 expression and glycolysis.TCGA analysis revealed a significant positive correlation between elevated HK2 expression and CD274 expression in patients with HCC (n = 360, Fig. 2a).Additionally, a similar association was observed with elevated SLC2A1 expression, which encodes glucose transporter protein type 1 (GLUT1; Fig. 2b).Further, the protein expression levels of PD-L1 and HK2 were verified using an HCC tissue microarray comprising 24 samples (Fig. 2c and Supplementary Table S1).A statistically significant positive correlation was observed between the expression levels of PD-L1 and HK2 (R 2 = 0.3878, p = 0.0080; Fig. 2d).However, the limited sample size precluded a statistically significant correlation from being established between PD-L1 and the TNM Classification of Malignant Tumors (TNM) stages.Overall, these findings confirm a positive correlation between PD-L1 expression and glycolysis in patients with HCC.

Upregulation of PD-L1 expression in sorafenib-resistant HCC cells
Sorafenib is a commonly used first-line chemotherapy for the treatment of advanced HCC; however, patients often develop drug resistance within 6 months 3 .To comprehend the correlation between PD-L1 expression, glycolysis, and sorafenib resistance, we employed sorafenib-resistant SK-Hep1 cells, which are induced through exposure to increasing concentrations of sorafenib (1-10 μM) over 6 months.(IC 50 of SK-Hep1 = 9.1 μM, IC 50 of SK-Hep1R = 18.9 μM, Supplementary Fig. S2).Glucose uptake increased in SK-Hep1R cells following IFN-γ treatment compared to that in corresponding SK-Hep1 cells (Fig. 3a), as well as the upregulation of HK2 expression (Fig. 3b).The expression of CD274 was upregulated in SK-Hep1R cells compared with that in SK-Hep1 cells (Fig. 3c).Similar to the RNA expression levels, PD-L1 in SK-Hep1R cells exhibited much higher expression than the SK-Hep1 cells at the protein level, while HK2 showed a marginal increase in SK-Hep1R cells (Fig. 3d,e).Furthermore, glucose treatment resulted in a significant elevation of PD-L1 levels in SK-Hep1R cells compared to SK-Hep1 cells (Fig. 3f,g).These results suggest that glucose deprivation is a potential strategy for reducing elevated PD-L1 expression in highly glycolytic, sorafenib-resistant HCC cells.

Regulation of PD-L1 expression following rapamycin treatment
Autophagy induction has been observed following glucose deprivation in cells 23 .Therefore, to regulate PD-L1 expression, rapamycin, an autophagy inducer utilized in several clinical trials for treating advanced HCC, was employed.Rapamycin treatment induced a reduction in PD-L1 and HK2 expression levels in HepG2 cells overexpressing PD-L1, similar to the effects of glucose deprivation (Fig. 4a,b).Notably, combined treatment with rapamycin (0.1 µM and 1 µM) and sorafenib (10 µM) resulted in the lowest PD-L1 expression in HepG2 cells with PD-L1 overexpression, compared to single treatments (Fig. 4c,d).The expression of HK2 after combination treatment showed levels lower than those observed with single sorafenib treatment, but not lower than rapamycin treatment.SK-Hep1R cells, treated with IFN-γ, also exhibited reduced PD-L1 and HK2 expression after rapamycin treatment.SK-Hep1R cells demonstrated a more pronounced reduction in PD-L1 and HK2 www.nature.com/scientificreports/compared to SK-Hep1 cells (Fig. 4e,f).Thus, rapamycin treatment might regulate PD-L1, which is amplified in sorafenib-resistant cells.

Enhanced anticancer activity following rapamycin treatment
To evaluate the PD-L1 and PD-1 binding affinity following rapamycin treatment, co-culture experiments were performed using Jurkat-Lucia™ TCR-hPD-1 and Raji-APC-hPD-L1 cells.Jurkat-Lucia™ TCR-hPD-1 cells were genetically engineered to produce a bioluminescent signal when the interaction between PD-L1 and PD-1 is inhibited.Relative light units (RLUs) increased after rapamycin treatment, indicating that rapamycin decreased the binding affinity between PD-L1 and PD-1 in the co-culture system (Fig. 5a).Since rapamycin reduced the binding of PD-1 and PD-L1, the cytolytic activity of Jurkat-Lucia™ TCR-hPD-1 cells was measured in co-culture with SK-Hep1R cells to examine the potential anticancer effect.Rapamycin enhanced the cytolytic activity of Jurkat-Lucia™ TCR-hPD-1 cells in these co-cultures (Fig. 5b,c).Compared with the control, the mRNA expression levels of the pro-inflammatory cytokines TNF-α, IFN-γ, and IL-2 were significantly increased in Jurkat-Lucia™ TCR-hPD-1 cells after co-culture with SK-Hep1R cells treated with rapamycin (Fig. 5d-f).In conclusion, rapamycin caused a reduction in PD-L1 levels and significant induction of T cell-mediated cytotoxicity against SK-Hep1R cells (Fig. 5g).www.nature.com/scientificreports/HCC 24,25 .Recent studies have supported the notion that elevated PD-L1 expression is associated with increased glycolysis in tumors, leading to the immune escape of tumor cells 26 .In the present study, we confirmed the positive correlation between PD-L1 expression and glycolysis in sorafenib-resistant HCC cells.We also established that rapamycin treatment enhances the cytolytic activity of immune cells by decreasing PD-L1 expression in sorafenib-resistant HCC cells.Sorafenib has been shown to improve survival outcomes in patients with HCC; however, its long-term efficacy is impaired by the emergence of resistance through various mechanisms.A recent study by Lu et al. identified elevated PD-L1 expression in tumor-infiltrating immune cells of patients with HCC following sorafenib treatment 18 .Our study further revealed the upregulation of CD274/PD-L1 in HCC cells upon acquisition of sorafenib resistance.The upregulation of PD-L1 aggravates sorafenib-resistant HCC cells by promoting epithelialmesenchymal transition through the PI3K/Akt pathway 27 .Liu et al. also demonstrated that the PD-L1/DNMT1 axis plays a role in sorafenib resistance in HCC, and inhibiting both PD-L1 and DNMT1 expression could restore the sensitivity of cells to sorafenib 28 .These previous studies lacked a clear understanding of why PD-L1 expression is increased in sorafenib-resistant cells.Our study revealed that sorafenib-resistant cells exhibit high glucose uptake and that this increase in glycolysis leads to elevated PD-L1 levels.Hypoxia, a major mechanism underlying glycolysis and cancer therapy resistance, induces PD-L1 upregulation in various tumor cells 29,30 .Therefore, hypoxia-induced glycolysis may contribute to the upregulation of PD-L1 and HK2 in patients with HCC.Hypoxia has been reported to be a major driver of resistance to cancer therapeutics 31 .
The FDA has approved the use of rapamycin for renal and liver transplantation.Patients with recurrent HCC display high levels of PD-L1 expression and reduced relapse-free survival compared with those with lower PD-L1 expression 19 .Recent studies have demonstrated a reduction in PD-L1 expression in non-small cell lung cancer lines following exposure to rapamycin, an autophagy inducer 32 .Our study shows that rapamycin treatment reduces PD-L1 expression, making it more effective against sorafenib-resistant cells.The interplay between autophagy and the PD-1/PD-L1 axis has been studied in various cancers.Wang et al. discovered that autophagy regulates PD-L1 expression in gastric cancer via the p62/SQSTM1-NF-κB pathway 33 .Further, in bladder cancer, PD-L1 mRNA stability, and expression are regulated by the ATG7/autophagy/FOXO3A/miR-145 axis 34 .Recent studies have reported the synergistic anticancer effects of a combination of sorafenib and rapamycin treatment on HCC cells.Gulhati et al. demonstrated that the addition of sorafenib to rapamycin treatment resulted in the abrogation of rapamycin-induced activation of the PI3K/Akt and Ras-MAPK signaling pathways, leading to enhanced antitumor effects 5 .Combination therapy significantly reduced tumor cell proliferation and increased the suppression of tumor cell angiogenesis compared to a single treatment in an in vivo animal model 35 .Additionally, our study highlights the potential anticancer effects of combining rapamycin therapy with sorafenib in HCC, with specific emphasis on the treatment of sorafenib-resistant HCC.Long-term treatment with other anticancer drugs has been shown to alter PD-L1 expression.High levels of PD-L1 have been observed in cisplatin-resistant small-cell lung carcinoma cells and enzalutamide-resistant prostate cancer cells 36 , suggesting the need for further investigation into the effects of rapamycin and other anticancer drugs on resistant cancer cells.
The overexpression of PD-L1 in various tumors is associated with patient survival and tumor recurrence.PD-L1 expression is a significant prognostic indicator of overall survival in HCC 37 .Additionally, PD-L1 regulates stem-like properties and contributes to tumor invasion.Thus, reducing PD-L1 expression, which is associated with tumorigenesis, is crucial for effective tumor treatment.

Conclusions
Our findings suggest that sorafenib resistance leads to increased PD-L1 expression through elevated glucose metabolism, resulting in decreased sensitivity to PD-1/PD-L1 inhibitor therapy.Collectively, the combination of rapamycin and sorafenib presents a new therapeutic option for counteracting the upregulation of PD-L1 expression in sorafenib-resistant HCC cells, leading to a reduction in tumor aggressiveness.www.nature.com/scientificreports/nonlinear regression analysis using GraphPad Prism (Supplementary Figure S2, IC 50 of SK-Hep1 = 9.1 μM, IC 50 of SK-Hep1R = 18.9 μM).

Cell viability and glucose uptake assays
A Cell Counting Kit-8 (CCK-8, DOJINDO Laboratories, Kumamoto, Japan) assay was performed according to the manufacturer's recommendations.To measure the binding affinity of PD-L1 for PD-1, Raji-APC-hPD-L1 cells (approximately 5 × 10 4 cells/mL) were pre-incubated with rapamycin for 6 h and co-cultured with Jurkat-Lucia™ TCR-hPD-1 cells (approximately 1 × 10 5 cells/mL) in a 96-well plate.After an additional 24 h of incubation, 50 µL of QUANTI-Luc™ 4 reagent (InvivoGen) was added to each well, and luminescence was immediately measured using a luminescence microplate reader (PerkinElmer, Waltham, Massachusetts, USA).To measure cytolytic activity, SK-Hep1R cells were maintained for 48 h at 37 °C in 12-well plates at an effector cell: target cell (E:T) ratio of 10:1.The cytolytic activity was assessed using crystal violet staining (0.25% v/v in phosphate-buffered saline (PBS)) after fixation with methanol.Positive tumor regions were quantified using cellSens Imaging software (Olympus, Tokyo, Japan), with images acquired using a BX53 light microscope (Olympus).Glucose uptake was determined using a Glucose Assay kit (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions.Absorbance was measured at 440 and 640 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

Statistical analysis
Statistical analyses were performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).One-way analysis of variance (ANOVA) with Tukey's multiple comparison tests was performed to detect differences between three or more groups.A paired two-tailed Student's t-test was used to detect significant differences between the two sets of data, and p < 0.05 was considered statistically significant.